Rubber is found in more than two thousand plant species, but is limited to only a few plant families (See Backhaus, Israel J. Bot., 34:283-293 (1985); Archer et al., Chemistry and Physics of Rubber-like Substances, Bateman, L. ed., pp 41-72, MacLaren, London (1963)). Rubber is a polymer composed of between 320-35,000 isoprene molecules. These are linked by stepwise, head-to-tail, cis-1,4 condensations that form the polyisoprene chains. The stepwise isoprene additions are performed by a prenyltransferase enzyme [E.C. 2.5.1.20] known as rubber transferase (RuT), rubber polymerase and rubber cis, 1-4 polyprenyl transferase. RuT had hiterto been considered to be the sole enzyme responsible for rubber formation in plants (See Backhaus, Israel J. Bot. 34: 283-293 (1985); Berndt, U.S. Government Res. Rep. AD-601, 729 (1963); Archer and Cockbain, Methods in Enzymology, 15:476-480, (1969); Archer and Audley, Advances in Enzymology, 29:221-257, (1967); and Lynen, Rubber Res. Inst. Malaya, 21:(4) 389-406 (1969)). However, according to the present invention it has been discovered that there is another enzyme, known as RPP (for rubber particle protein) that clearly causes polyisoprene biosynthesis when transferred to a foreign host plant, such as tobacco. As set forth herein, RPP, unlike RuT, is not a prenyltransferase but rather functions as an allene oxide synthase (AOS). RPP has properties similar to other members of the class of heme-binding enzymes to which AOS belongs (e.g. cytochrome P450) and is an enzyme needed for rubber biosynthesis. P450 enzymes have never before been implicated in rubber biosynthesis.
The first identification of guayule RPP was made in 1985 (Backhaus and Chandra, in Alcorn, S. and Fangmeier, D. (eds) Proceedings of the 4.sup.th International Guayule Conference on Guayule Research and Development, October 16-19, (1985); Backhaus and Bess, in Randall, D. D. et al. (eds) Current Topics in Plant Biochemistry and Physiology, 5:186 (1986)). Subsequent publications described its purification and characterization and suggested a putative role as a prenyltransferase in guayule rubber particles (Backhaus and Bess, in Benedict, C. R. (ed) Biochemistry and Regulation of cis-Polyisoprene in Plants, NSF sponsored workshop publication, p. 204-220 (1986); Cornish and Backhaus, Phytochem. 29:3809-3813 (1990); and Backhaus et al. Phytochem. 30:2493-2497 (1991)). Work by Cornish and Siler demonstrated that RPP-like proteins were also present in Hevea and Ficus (Siler and Cornish, Phytochem. 32: 1097-1102 (1993); Cornish, et al. J. Nat. Rubb. Res. (1994); Cornish, et al., Phytochem. (1994); Siler and Cornish, Phytochem. (1994)). The RPP-like proteins in rubber particles of these other species are likely involved in the same enzymatic reaction as RPP. Reference to another protein isolated from guayule rubber particles was made (Benedict et al., Plant Physiol. 92:816-821 (1990)) which described a prenyltransferase but did not identify that protein as RPP despite prior knowledge by those authors (cf. Benedict, C. R. (ed) Biochemistry and Regulation of cis-Polyisoprene in Plants, NSF sponsored workshop publication, p. 204-220 (1986)) of RPP's existence in guayule rubber particles.
RPP is a membrane glycoprotein having an isoelectric point (pI) of 6.2 (Backhaus et al., Phytochem. 30:2493-2497 (1991)). Prior to the present invention, the molecular weight of RPP was estimated to be 48,500 to 53,000 Daltons as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). RPP is the most abundant protein of guayule rubber particles (Backhaus et al., Phytochem. 30: 2493-2497 (1991)) and was, therefore, considered to be important for rubber biosynthesis.
Rubber, like polyunsaturated fatty acids (PUFAs), is extremely susceptible to autoxidation by free radicals due to the presence of a double bond in each per 5 carbon isoprene monomer (Brydson, Rubber Chemisty, Applied Science Publishers, Inc., Essex (1978)). In rubber, peroxidation changes the physical properties of elastomers with the net effect being chain scission, and/or cross-linking of polymers. In addition, the formation of volatile oxidation products can also occur (Shelton, Rubber Chem. Tech. 45:359-377 (1972)). For each molecule of peroxide on polyisoprene, one cross-link can be formed (Shelton, Rubber Chem. Tech. 45:359-377 (1972)). In addition, rubber particles contain PUFAs, which are also susceptible to peroxidation and can initiate autoxidation of rubber. An antioxidant effect would therefore be beneficial to rubber synthesis.
There are many ways in which an antioxidant effect may be beneficial to diverse biological systems. Lipid peroxides and hydroperoxides are responsible for numerous disorders, including senescence and aging. Lipid peroxidation in biological systems is a consequence of normal metabolic processes in a cell. Active oxygen species such as hydrogen peroxide and superoxide are generated by free radical mediated processes. These species attack susceptible polyunsaturated lipids to form lipid peroxides, which in turn initiate the peroxidation of adjoining lipids in a chain reaction process. Lipid hydroperoxides are also formed enzymically by lipoxygenase (LOX).
Organisms have developed a number of strategies to overcome the damaging effect of these oxidative attacks. One set of mechanisms involves the production of small antioxidant molecules which scavenge free radicals (i.e. beta carotene) or intervene in the formation of lipid peroxides by a chemical reduction (i.e. alpha tocopherol) to break the chain propagation steps. Cells producing adequate amounts of these compounds keep the lipid peroxides from causing cell damage. Another mechanism involves the production of antioxidant enzymes which disrupt various steps of oxidative reactions. Among the enzymes currently known to be involved in these processes are superoxide dismutase (SOD), catalase, ascorbate perioxidase and glutathione peroxidase. For example, the over expression of chloroplast-activated Cu/Zn superoxide dismutase (SOD) acts as an antioxidant, increasing the resistance to oxidative stress in transgenic tobacco plants (Gupta et al. Proc. Nat'l Acad. Sci. USA 90:1629-1633 (1993)). Gupta used a cell leakage analysis to measure cellular damage of leaf disks of transgenic SOD and untransformed cv. Xanthi plants by measurement of the percent solute leakage in treated tissues compared with autoclaved tissues.
Antioxidants, such as beta-carotene, SOD, allopurinol, and catalase, have been used in treating ischemia following surgery or injury in biological organisms. (Konovalova et al., Arkh. Patol. 51(6): 19-24 (1989) (Abstract); Minor et al., Surg. Today 23(8): 728-732 (1993) (Abstract); Maksimenko et al., Eksp. Kin. Farmakol. 56(5): 14-18 (1993) (Abstract); Kloner, Circ. Res. 64:(1): 86-96 (1989) (Abstract); Nayak et al., Invest. Ophthalmol. Vis. Sci. 34(6): 2018-2022 (1993) (Abstract); Baker et al., Ann. Surg. 202(5): 628-641 (1985) (Abstract); Gross et al., Am. J. Physiol. 250(3Pt.2): H372-H377 (1986) (Abstract); Zweier et al., J. Clin. Invst. 80(6): 1728-1734 (1987) (Abstract); Singh et al., Mol. Cell. Biochem. 125(2): 97-104(1993); Nelson et al., Free Radic. Biol. Med. 16(2): 195-200 (1994) (Abstract); Burton, Am. J. Physiol. 248(5 Pt. 2): H637-H643 (1985) (Abstract); Chi et al. Circ. Res. 64(4): 665-675 (1989) (Abstract); Miura et al. Jpn. Circ. J. 53(7): 786-794 (1989) (Abstract); Konovalova et al. Biull Eksp Biol. Med. 98(8): 153-156 (1984) (Abstract); Gutkin et al. Biull. Eks. Biol. Med. 93(1): 33-35 (1982) (Abstract); and Wang et al., Hua. Hsi. I. Ko. Ta. Hsueh. Pao. 25(1): 62-65 (1994) (Abstract)). Ischemia is a deficiency of blood in a biological organism due to functional constriction or actual obstruction of a blood vessel, common in surgery, that causes cell tissue damage as a result of oxidation. Myocardial ischemia is a deficiency of blood supply to the heart muscle.
It is known to the art that the over expression of antioxidant enzymes retards the age-related accrual of oxidative damage. By the over expression of superoxide dismutase in Drosphilia melanogaster, the average and maximum life-span of transgenic fruit flies is extended. (Sohal et al., Science 273: 59-63 (1996)).
In contrast to the present invention, none of the above-cited references teach the use of an allene oxide synthase (such as RPP) as an antioxidant and none teach a beneficial curative effect to reduce or prevent cell damage caused by oxidative attacks.